The present invention is based on scientific analysis of mRNA data from several samples of human cord blood and cross analysis between data from different sources. The mRNA data is also analyzed with regard to large amount of scientific literature about expression of proteins or mRNA in other related systems.
Preferred General Cell Types
The present invention is specifically directed to human cells, specifically to human cord blood cells. It is realized that use of mRNA and protein vary between species, so that making reliable parallels about exact roles of molecules in different species may not be possible. The present invention is preferably directed to native cells, meaning non-genetically modified cells. Genetic modifications are known-to alter cells in unpredictable ways, the unexpected changes in context of genetic transfection would require very broad control with regard to numerous biochemical systems existing in cells. The present invention is further directed in a preferred embodiment to fresh cells meaning non-cultivated cells as a cell population to be profiled by the method according to the invention.
Generally Non-Useful Bulk Genomics and Other Database Data or Combinations Thereof with Regard to Present Invention
The inventors realize that data may already exist about now listed mRNA markers from various cell populations. However, this data do not necessarily indicate the usefulness of the data for any analytical method, since the amount of the data may be enormous and thus the usefulness of any specific single marker may be impossible to indicate. It is further realized that retrospective combinations of the database data does not allow determining usefulness of a marker, because such combinations would reveal endless number of targets for various uses without specific analysis and data to support the real usefulness of the data. It is further realized that single data point data include experimental variations and risks to such level that the scientist publishing the study may have not considered it useful for specific commercial application as such.
Preferred Markers and Marker Groups with Regard to Preferred Cell Populations
Hematopoietic Stem Cell Related Marker
The preferred hematopoietic stem cells related-markers include CD133, CD34, KIT, TIE1(TIE), ANGPT1, SCA-1, and MEIS1.
Background Related to ANGPT1
Angiopoiesis regulated genes has been studied in cord blood CD34+ cells. Potentially angiopoiesis related mRNAs TIE1, Ang-1 and Ang-2 have been detected enriched in doubly selected cord blood cell populations CD133+/CD34+ when compared to CD133−/CD34− cell line (Pomyje J. et al. Eur J. Haematol. (2003) 79 (3) 143-50). This work does not define the difference of the mRNA expression between CD34+ or CD34−, CD133+ and CD133− cells and other cell populations.
The present invention is specifically directed to ANGPT1 as a marker of CD133 positive complete cell populations, preferably complete blood derived cell populations, more preferably complete cell populations derived from cord blood.
TIE1
Presence of TIE1, among several other markers has been noted in magakaryocytes and CD34+ progenitor cells from cord blood (Blood (1996) 87 (6) 2212-20, Batard P et al.), it is further indicated to be associated with marker Flt3 (Blood (1997) 90, 111-125 Rappold, I et al.).
The present invention shows TIE1 as an important marker of CD133 cell populations and of complete cord blood cell populations, especially from complete CD133 cell populations and in combination with other important markers.
SCA-1
SCA-1 has been reported to be present in murine sinusoidal endothelial cells, it is also noted as a murine hematopoietic stem cell marker (Luna, G et al. Stem Cells Dev. (2004) 13 (5) 528-35; Liu Y J. Et al Blood (2004) 103 (12) 4449-56). In a review of both human and murine hematopoietic stem cells sca-1 and c-kit were indicated as marker of murine cells (Wognum A W. Et al. Arch. Med Res. (2003) 34 (6) 461-75).
The present invention is preferably directed to markers of human cells for which the background is not relevant. SCA-1 is preferred for all cells and methods according to the invention, most preferably for the preferred CD133 type cells.
MEIS1
This transcription factor has been reported to control expression of CD34 and FLT3 in human leukemia cells (AML) immortalized by Hoxa9 (Wang G G et. al. Blood (2005) Mar 8, publ. Ahead print). MEIS1 has been further detected in megakaryotes differentiated from CD34+ cord blood cells.
The present invention is preferably directed to native cells, meaning non-genetically modified cells, the present invention further directed in a preferred embodiment to fresh non-cultured cells.
MEIS1 is a preferred angiogenesis, especially capillary angiogenesis associated marker. MEIS1 is preferred for all cells and methods according to the invention, most preferably for the preferred CD133 type cells.
KIT (CD117)
KIT receptor tyrosine kinase has been reported from CD34+ cord blood cell populations by monoclonal antibodies 80-95% coexpressed with FLT3 (Rappold I et al. Blood (1997) 90 (1) 111-25). In another study CD34+ Kit+ and -cells were used in a transplantation model (Tanabe Y et al. BBRC (2004) 324 (2) 711-8). Kit protein expression was observed in a CD133 positive cord blood cell preparation but it was not indicated as an over expressed marker or otherwise useful marker (Ruziska K et al. (2004) Clin Chim. Acta. 343 (1-2) 85-92). In a review of both human and murine hematopoietic stem cells sca-1 and c-kit were indicated as marker of murine cells, and CD133 for human cells (Wognum A W. Et al. Arch. Med Res. (2003) 34 (6) 461-75).
The present invention shows KIT as an important marker of complete and/or homogenous cell populations, especially from complete, especially human, CD133 cell populations and in combination with other important markers.
Preferred Gene Clustering Methods Derived Markers
Three groups of similarly expressed markers were obtained. The preferred markers includes SPINK2, CD133, CD34, KIT, FLT3, LAPTM4B, EBPL, CRIM1, ANKRD28, DKC1, BAALC and JUP. The preferred subgroups are    group1: SPINK2, CD133, CD34, and KIT, more preferably SPINK2 and KIT, most preferably SPINK2;    group2: FLT3, LAPTM4B, EBPL and CRIM1;    group3: ANKRD28, DKC1, BAALC and JUP.Ankyrin Repeat Domain 28, ANKRD28
ANKRD28 was cloned from human brain with no indication of function (Nagase T et al. DNA Res. (1997) 4 (2) 141-50). ANKRD28 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
Baalc (Brain and Acute Leukemia Cytoplasmic)
Baalc has been reported in another context as development related protein from CD34+ bone marrow cells (Baldus, C D et al. (2003) 31 (11) 1051-6) and to be associated with mesoderm and muscle development (Satoskar A A et al Gene Expr Patterns (2005) 5 (4) 463-73).
Baalc (brain and acute leukemia cytoplasmic) mRNA appears to be novel marker for the preferred cell populations according to the invention.
Dyskeratosis Congenita 1 Dyskerin, DKC1
Mutation in DKC1 (dyskerin causes) rare X-linked form of disease dyskeratosis cognita, characterized with mucocutaneous abnormalities and bone marrow failure, premature ageing and a predispoposition for malignancy, patients have very short telomeres (Vulliamy T J et al. Blood molecular dis. (2001) 27 (2) 353-7). The disease resembles aplastic anemia and myeloplastic syndrome, stem cell transplantation may be a treatment (Bessler M et al. Curr Opin Pediatr. (2004) 16 (1) 23-8.
DKC1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
EBPL Emopamil Binding Protein Like
EBPL, emopamil binding protein-like has been indicated for sterol synthesis and X-chromosomal chondrodysplasia punctuata (Moebius F F, et al. Biochem J 374 229-37). EBPL mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
Serine protease Inhibitor, Kazal Type 2 (Acrosin-Trypsin Inhibitor), SPINK2
SPINK2 has been associated with defects in sperm development. SPINK2 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
Cell Adhesion Related Molecules
The invention is further specifically directed to 12 mRNA-markers that encode adhesion related molecules and were up-regulated in CD133+ cells. The overexpression of these genes (CD34, COL5A1, DSG2, DST, IL-18, ITGA9, JUP, PKD2, SEPP1, TRO, VAV3, and VLA-4) was observed in CD133-type cells according to the invention. The invention is preferably directed to individual novel markers and subgroups thereof, the individual novel markers according to the invention includes COL5A1, DSG2, DST, IL-18, ITGA9, JUP, PKD2, SEPP1, TRO, VAV3, and VLA-4.
Part of the markers are especially preferred as cell adhesion supportive factors, especially in connection with cell junctions and cytoskeleton, these are referred as group 1 (of cell adhesion related molecules): DSG2, DST, JUP, PKD2, VAV3. Part of the molecules have roles as extracellular proteins supporting cell adhesion, referred under the embodiment as group 2, such as IL-18, COL5A1, SEPP1, The third preferred group includes cell adhesion receptors ITGA9, and VLA-4.
IL-18
IL-18 is cell adhesion related protein but as a cytokine not a direct cell adhesion receptor molecule.
High levels of IL-18 in neonatal cord blood have been associated with periventricular leukomalacia (neonatal white matter damage) which often leads to cerebral palsy (Minaga K. et al. (2002) 17 (3) 164-70).
The background data do not indicate expression of IL-18 by human cord blood cells, especially by the preferred subpopulations such as CD133+ cells, and for stem cell populations in general.
JUP (Plakoglobin, Gamma-Catenin)
Plakoglobin is expressed on protein level throughout zebrafish embryo development and it is observed in desmosomes during heart chamber development (Martin and Grealy (2004) 32 (15) 797-9). The protein is required for embryonic heart, neuroepithelium, skin and vasculature (Galliciano G L et al (2001) 128 (6) 929-41). JUP mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
JUP may function as intracellular protein and its role is especially related to cell junctions and support of cell adhesion.
Polycystin-2 PKD2
PKD2 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
PKD2 may function as intracellular protein and its role is especially related to cell junctions and support of cell adhesion.
VAV3
VAV1-3 knock out indicate that especially VAV1 but also others are important for B and T-lymphocyte function (Fujikawa K et al. (2003) 198 (10) 1595-608). VAV3 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
VAV3 may function as intracellular protein and its role is especially related to cell junctions and support of cell adhesion.
VLA4, (Very Late Activation Antigen 4), Integrin Alpha4Beta1
Several publications have indicated the presence of VLA4 in CD34 positive cord blood populations. The data do not indicate its presence in context of other preferred cell related adhesion molecules except CD34.
VLA4 has been further been analyzed in CD34 cell populations in comparison to CD133 cord blood population in cell growing system as a potential cell adhesion molecule. VLA4 was not analyzed to be a specifically enriched marker in native CD133+ cells but these were indicated as base line expression. The data of the abstract does not indicate usefulness of VLA4 as overexpressed specific marker in CD133 cells (Zhai Q L et al. Zhongguo Yi Xue Ke Xue Yuan Xue Bao (2002) 24 (1) 7-10.)
Preferred Receptor Molecules
The inventors revealed markers related to preferred receptor molecules. These includes preferred cell adhesion receptors (group 1 under the embodiment), regulatory receptors (group 2) and growth factor receptors (group 3).
Preferred cell adhesion receptors include ALCAM (Activated leukocyte cell adhesion molecule), ITGA9, and VLA-4.
Preferred growth and activating factor receptors include CRIM1 (cysteine-rich motor neuron 1), FLT-3, SCA-1, KIT, TIE1, LRP6 Low density lipoprotein receptor-related protein 6, TNFRSF21 (tumor necrosis factor receptor superfamily, member 21)
Preferred regulatory receptors include: PTPRD (Protein tyrosine phosphatase, receptor type, D), PILRB (paired immunoglobin-like type 2 receptor beta), ADAM28.
Soluble Regulatory Proteins Including Growth Factors and Cytokines
The inventors were able to define certain growth factors and cytokines being specifically associated with the preferred cell populations.
The invention is especially directed to growth factor and cytokine markers according to the invention including:    ANGPT1,    AREG Amphiregulin (schwannoma-derived growth factor),    IGFBP7 (Insulin-like growth factor binding protein 7,            Angiomodulin/Mac25/tumor adhesion factor TAF,        Insulin-like growth factor binding protein-related protein 1),            IL-18,    soluble ALCAM
The invention is further directed IGFBP7 (Insulin-like growth factor binding protein 7) as a special growth factor regulating protein.
Cell Matrix Related Markers
The invention is further specifically directed to cell matrix related markers    col5a1, Collagen type V alpha 1, col5a1    MMP28, matrix metalloproteinase 28LAPTM4B
Expression of this mRNA has been reported from certain embryonal stem cell lines (Abeyta Hum. Mol Genet. 2004; 13:601-608), from HSCs and neuronal stem cells (Ivanova N B. Et al. Science. 2002; 298:601-604; Ramalho-Santos M. et al. Science. 2002; 298:597-600) and from hepatocellular carcinoma tissues (Liu X R. Et al World J Gastroenterol. 2004; 10:1555-1559).
The mRNA has not specified as a specific marker of the cell populations according to the present invention.
Transmembrane and/or Membrane Associated Proteins
The present invention is preferably directed to following markers related to transmembrane and/or membrane associated proteins, the corresponding Affymetrix probe set ID is indicated in Table 2:
ADAM28
ADAM28 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
ALCAM, Activated Leukocyte Cell Adhesion Molecule (Mouse Dm-Grasp Protein; Rat MEMD protein, HB2, SB-10 Antigen, KG-CAM)
ALCAM has been indicated in context of mesenchymal stem cells. Prior art does not indicate ALCAM in cord blood, or in preferred cell populations derived from it nor in CD133 cells. ALCAM mRNA appears to be novel marker for the preferred cell populations according to the invention.
Amphiregulin AREG
Actually soluble glycoproteins growth factor at least in most cases, it is preferred also as a membrane associated protein, larger isoform is known.
AREG has been described from cord blood derived cultivated mast cells, but not from preferred cell populations according to the present invention (J Allergy Clin Immunol (2005) 115 (2) 287-94 Wang S w et al.).
AREG mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
ATP9A (ATPase, Class II, Type 9A)
ATP9A mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
CRHBP/CRH-BP, Corticotropin-Releasing Hormone-Binding Protein/ CRFBP/CRF-BP, Corticotropin-Releasing Factor-Binding Protein/
CRHBP mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
CRIM1
Crim1 expression has been shown from early condensing mesenchyme and distal comma shaped bodies of uretheric tree, and male-specifically from Sertoli cells of developing testis, being yet another spermatogenesis associated protein (Georgas K et al. (2000) 219 (4) 582-7). Crim1 gene expression has been further detected in endothelial of forming capillary structures and in endothelial cells of inner lining of blood vessels. The corresponding protein was shown to be glycosylated and accumulate close to cell-cell contacts (Glienke J et al. (2002) 199 (2) 165-74). Crim1 has been also studied from chick spinal cord (Kolle G. et al. Dev. Dyn (2003) 226(1) 107-11) and in murine ocular development (Lovicu F J et al. (2000) 94 (1-2) 261-5).
CRIM1 mRNA appears to be novel marker for the preferred human cell populations according to the invention, and for human stem cell populations in general.
C14rf1 Chromosome 14 Open Reading Frame 1
C14orf1 may be indicated in a CD34+ cultivated immortalized cancer cell lines having no relevance with regard to present cells (Genome Res. (2000) 10, 1546-60 Zhang Q-H et al.). C14orf1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
CYYR1 (Cysteine and Tyrosine-Rich 1)
CYYR1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
Desmoglein 2, DSG2
DSG2 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Epithelial Membrane Protein 1, EMP1
EMP1 belongs to peripheral myelin protein 22 (PMP22) family, it has been indicated from early neurons during neuritogenesis in mouse brain and in neuroectodermally differentiated P19 cells (Brain Res Dev Brain Res (1999) 166 (2) 169-80 Wulf and Suter).
EMP1 mRNA appears to be novel marker for the preferred human cell populations according to the invention, and for human stem cell populations in general
FLT3
FLT3 is a receptor tyrosine kinase binding flt-3 ligand, which is a growth factor type molecule used in hematopoietic cell cultures. A few background articles notes use of flt-3 ligand for proliferation of CD133+ cells from cord blood, this does not indicate FLT3 expression or it as a useful marker overexpressed in context of CD133, expression levels or it change were not indicated, the studies were directed to cultivated expanded cells and not to primary cell preferably directed by the invention (Thromb haemost. 820049 92 (4) 767-75 Baal N et al.; Stem Cells (2004) 22 (1) 100-8 Forraz N et al; Haematologia (2003) 88 (4) 388-95 Encabo A. et al; Transfusion (2003) 43 (3) 383-9 Encabo et al; J Heatother. Stem Cell Res. (2001) 10 (2) 273-81 Kobari L et al.; Cell Prolif. (2004) 37 (4) 295-306, McGuckin C P et al.). FLT3 has been widely studied in context of cord blood cells. It has been also used for selection of cell populations from cord blood. Specific overexpression in cord blood CD133+ cells has not been indicated.
FLVCR (Feline Leukemia Virus Subgroup C Cellular Receptor)
FLVCR mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
GPR125 (G Protein-Coupled Receptor 125)
GPR125 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
IGFBP7 (Insulin-Like Growth Factor Binding Protein 7)
IGFBP7 mRNA and protein has been indicated from bone marrow stromal stem cells, implied as osteoprogenitor cells, and dental pulp stem cells (Shi, S et al. (2001) Bone 29 (6) 532-9). IGFBP7 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Integrin 9Alpha, Alpha9Beta1 Integrin, ITG9A
ITG9A mRNA appears to be novel marker for the preferred cell populations according to the invention.
KIAA0286
KIAA0286 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
KIAA0152
KIAA0152 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
LRP6
LRP6 mRNA appears to be novel marker for the preferred cell populations according to the invention.
MMP28 Matrix Metalloproteinase 28
The invention is preferably directed to MMP28 as a membrane associated marker.
MMP28 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
PILRB (Paired Immunoglobin-Like Type 2 Receptor Beta)
The protein has shown to be overexpressed in erythroid progenitor cells in comparison to bone marrow released peripheral blood CD34+ cells.
PON2, Paraoxonase2
PON2 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
PTPRD (Protein Tyrosine Phosphatase, Receptor Type, D)
The literature background indicate absence of PTP delta from embryonic stem cells (Mol. Biol. Reprod. (1994) 19 (2) 105-8 Hendriks W et al.), absence of the type of PTP was further indicated from cultivated keratinocyte cell lines (J. Invest Deramtol. (1996) 106 (5) 972-6 Hendriks W et al.) PTPRD mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
SEPP1 (Selenoprotein P, Plasma, 1)
SEPP1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
The invention is specifically directed to SEPP1 as a membrane associated marker.
SLC16A14 (Solute Carrier Family 16 (Monocarboxylic Acid Transporters), Member 14)
SLC16A14 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
SV2A
SV2A mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
TM7SF3 (Transmembrane 7 Superfamily Member 3)
TM7SF3 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
Transmembrane 6 Superfamily Member 2 Isoform 2 TM6SF1
TM6SF1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
TNFRSF21 (Tumor Necrosis Factor Receptor Superfamily, Member 21)/Death Receptor 6, DR6
TNFRSF21 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Trophinin, TRO
TRO mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
Vezatin
Vezatin mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
Transmembrane and/or Membrane Associated Glycoproteins
The preferred mRNAs corresponding to potential N-glycoproteins preferably include:
AREG, ALCAM, ITGA9, FLT3, PTPRD, TM7SF3, PON2, DST, ADAM28, CRHBP, DSG2, EMP1, FSTL1, GPR125, IGFBP7, KIT1, MMP28, SCA-1, SV2A, SEPP1, TIE1, TNFRSF21, LRP6, OPN3
MRNAs of Possible Extracellular Proteins
The present invention is further directed to mRNA potentially corresponding to secreted cell regulating marker structures.
Following mRNAs are also preferred as marker related to secreted glycoproteins:    CRHBP/CRH-BP, corticotropin-releasing hormone-binding protein    IGFBP7 (Insulin-like growth factor binding protein 7).    MMP28 is a secreted protein, which may exist in membrane associated form SEPP1 (selenoprotein P, plasma, 1),    Uromodulin-like 1, orCollagen type V alpha 1, col5a1, A preferred matrix protein
Col5a1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Uromodulin-like 1, UMODL1
Present invention is specifically directed to UMODL1 mRNA, because the related protein uromodulin is strongly glycosylated protein with potential glycosylation associated functions. UMODL1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
LT-Markers (Lrp6/Tcf7L2-Related) and Cell Junction-Type Signaling Related Marker Groups
The preferred LT-markers includes Lrp6, γ-catenin (JUP, plakoglobin), Tcf7L2/TFC4, HOXA9, HOXA10, MAP3K4 and IL-18, more preferably Lrp6, γ-catenin (JUP, plakoglobin), Tcf7L2/TFC4, MAP3K4 and IL-18. The background related material of markers other than HOXA9 and HOXA10 is represented with other groups bellow or above (transcription factors, cell adhesion IL-18).
HOXA9 and HOXA10
HOXA9 and -10 have been reported from cord blood CD34+ cells (Ferrell C M et al. (2005) Stem Cells 23 (5) 644-55). These appear to be novel markers for the preferred cell populations according to the invention.
Transcription Factors
The present invention is further directed markers related to specifically expressed transcription factors.
STAT5
The transcription factor has been implicated in connection with hematopoietic differentiation of cord blood cells (Buitenhuis M et al. (2003) Blood 101 (1) 134-43).
Pubmed search did not indicate CD133 association.
The invention is specifically directed to the marker of STAT5 for analysis of CD133-type cell populations, and/or from complete cell populations according to the invention, derived from mononuclear cells and/or cell populations of human cord blood.
GATA-2
This transcription factor has been indicated in some cord blood derived CD34+ cell preparations (Pan X et al. (2000) 127 (1) 105-12), GATA-2 has been also indicated in vasculogenesis from human embryonal stem cells (Gerecht-Nir S et al. (2005) Dev. Dyn. 232 (2) 487-97).
The invention is specifically directed to the marker of GATA-2 for analysis of CD133-type cell populations, and/or from complete cell populations according to the invention, derived from mononuclear cells and/or cell populations of human cord blood.
Tcf7L2/TFC4
This signaling pathway molecule was not found to be indicated for CD34+, CD133+ or human cord blood cells in PubMed.
HOXA5
In a background publication transfection of CD34+ cord blood cells with HOXA5 was used in studies of hematopoiesis indicating suppression of erythropoiesis and promotion of myelopoiesis (Crooks G M et al. (1999) 94 (2) 519-28). HOXA5 gene expression has not been indicated as specifically overexpressed marker of early human cells, especially preferred cell populations according to the present invention.
Mitogen-Activated Protein Kinase Kinase Kinase 4, MAP3K4
MAP3K4 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Cell Cycle Related Markers
GATA2, N-MYC, DST, PLAGL1, NME1, CDK6, BCAT1, CDK4, BMI-1 MCM2, MCM5, MCM6, MCM7, CDK2AP1, SH3MD2, UHRF1, ZNRF1, EDD, SKB1, STAG1, ANAPC7 and MPHOSPH9. The invention is preferably further directed down regulated markers p18, and CDKN2D. Certain levels of cell cycle related markers are observed in connection with normal cell cycle in many types of cells. The present invention is in a preferred embodiment directed to analysis one or several, preferably at least 2, even more preferably at least 3 cell cycle related markers according to the invention in context of analyzing one or several other markers according to the invention. The analysis of part of the markers may be novel as such from the preferred cell types.
GATA2 is also preferred as a preferred transcription factor.
N-MYC
N-MYC mRNA expression has been reported from human fetal blood Lin-CD34+CD38− cells [Shojaei F et al. Blood (2004) 103 (7) 2430-40]. GATA2 and N-MYC showed 2-3 times higher levels of expression in peripheral blood than in bone marrow CD34+ cells, (Steidl U et al. Blood (2002) 99 (6) 2037-44). Pre-B cells do not have overexpressiion of N-Myc (Pathobiology (1992) 60 (2) 87-92, Wetherall And Vogler). Pubmed search did not indicated CD133 or cod blood association.
Dystonin DST
DST mRNA appears to be novel marker for the preferred cell populations according to the invention.
Pleiomorphic Adenoma Gene Like1, Plagl1 (Lot1/Zac1)
Plagl1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
NME1
NME1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cell populations in general.
CDKN2 (p16INK4a)
CDKN2 mRNA appears to be novel marker for the preferred cell populations according to the invention.
CDK4, Cyclin Dependent Kinase 4
CDK4 has been reported from cord blood mononuclear cells. TGF-beta1 inhibits its expression. This work does not indicate homogenous cell populations according to the invention (Zhonggou Shi Xan Xue Xe Xue Za Zhi (2004) 12 (5) 644-8 Shi B et al.). CKD4 has been indicated to be expressed, while other cyclins are not expressed in CD34+ bone marrow cells, status of cdk4 during hematopoiesis was no induction or stable expression, thus further indicating no specific analytical change with the cell populations quite different from the preferred cells according to the invention (Leuk Lymphoma (2002) 43 (2) 225-31 Furukawa Y et al.; Blood (2001) 97 (9) 2604-10, Lewis J L et al).
CDK4 mRNA appears to be novel marker for the preferred cell populations according to the invention.
CDK6
CDK6 has been reported from CD34+ hematopoietic cell lines and other hematopoietic cells (BBRC (1998) 231 (1) 73-6 Della Regione F et al.)
CDK4 mRNA appears to be novel marker for the preferred cell populations according to the invention.
CDK2AP1 (p12DOC-1)
CDK2AP1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
BCAT-1
BCAT-1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
BMI-1
BMI-1 has been reported from hematopoietic type cells developed from embryonic stem cells (Zhonggou Shi Xan Xue Xe Xue Za Zhi (2005) 13 (2) 222-8 Wang J et al.) and from CD34+ bone marrow cells with high expression in comparison to negative counterpart (Blood (1998) 91 (4) 1216-24 Lessard J et al. BMI-1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Minichromosome Maintenance Protein-2, MCM-2
MCM-2 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
Minichromosome Maintenance Protein-5, MCM-5
MCM-5 mRNA appears to be novel marker for the preferred cell populations according to the invention.
The invention is specifically directed to the mRNA for analysis of mononuclear cells and/or cell populations from human cord blood, preferably CD133-type cell populations. The present invention is further directed to MCM-5 mRNA as a novel hematopoietic stem cell marker.
Minichromosome Maintenance Protein-6, MCM-6
MCM-6 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
Minichromosome Maintenance Protein-7, MCM-7
MCM-7 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
Anaphase Promoting Complex Subunit 7, ANAPC7
ANAPC7 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
M-Phase Phosphoprotein 9, MPHOSPH9
MPHOSPH9 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
Ubiquitin-Like, Containing PHD and RING Finger Domains, 1, UHRF1
UHRF1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
SH3 Multiple Domains 2, SH3MD2
SH3MD2 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
Skb1
Skb1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
ZNRF1
ZNRF1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
STAG1/Stromal Antigen 1, Stro-1
Stro-1+ mesenchymal cells direct in NOD/SCID mouse to spleen muscles, BM, and kidneys while more Stro-1− directed to lungs. The cultivated mesenchymal cell populations were derived from cord blood but usefulness as a marker was not indicated nor presence in a homogenous cell populations, the cell may be different from CD34+ cells (Blood (2004) 103 (9) 3313-9 Besidhoum M et al.). In a preferred embodiment the invention is directed to primary cell populations without artificial cultivation or transfections.
Stro-1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
EDD
EDD mRNA appears to be novel marker for the preferred cell populations according to the invention.
p18
Cyclin Dependent kinase inhibitors p18INK4c was reported to be highly expressed in CD34+ progenitor cells and in acute myeloid leukemia cells, but not in normal myeloid cells (Br J hematol. (1999 106 (3) 644-51 Tschan, M P et al).
p18 mRNA appears to be novel downregulated marker and cancer related marker for the preferred cell populations according to the invention.
Potential Cell Migration Function Related Markers
The present invention is specifically directed to markers selected from the group: SPINK2, CD133 and SEPP1; more preferably SPINK2 and SEPP1 and most preferably SPINK2; as potential cell migration associated markers. These molecules have been associated with sperm/microvillus development. The invention is directed to the use of the marker SPINK2 together with any other preferred markers for methods according to the invention, more preferably together with CD133 and/or SEPP1.
Markers Related to Potential Endothelial Development
The present invention is specifically directed to markers according to the present invention when these have connection to potential proteins associated with potential endothelial development. The markers potentially related to endothelial development include ADAM28, ANGP1, CRIM1, DSG2, EMP1, JUP, MAGI1, TIE1.
Preferred potential endothelial development associated mRNAs were selected by comparing expression between CD133+ and CD34+, the following marker were revealed to be associated with CD133+ type cells ANGP1, DESG2, CRIM1, EMP1, ADAM28, and MAG1.
Association preferred emdothelial development related markers with CD133-type cells.
The table indicates expression fold change in comparison with corresponding negative cell population.
Mean foldMean foldchangechangeCD133+CD34+ANGPT118.79.6DESG210.76.5CRIM14.81.8EMP15.74.2ADAM114.45.5MAGI113.75.5MAG1, membrane associated guanylated kinase inverted-1MAGI1 mRNA appears to be novel marker for the preferred cell populations according to the invention.ESC-Related Stem Cell Markers
The present invention is specifically directed to transcriptional analysis of embryonal stem cell related markers in the preferred cell populations. Preferred ESC-related stem cell markers, in a preferred embodiment are DNMT3B, DNMT3A, and DPPA4.
DNMT3B and DNMT3A
Background about DNMT3B and DNMT3A describes hematopoietic cells from murine fetal livel, yolk sac, and adult bone marrow. Due to difference between species and tissues this is not relevant with regard to present invention (Gene Epr. Patterns (2004) 5 (1) 43-9, Watanab D et al). Another study describes human leukemia and bone marrow cells which express DNMT3B, DNMT3A, but human cord blood, such as cord blood markers were not described (Blood (2001) 97 (5) 1172-9 Mizuno S et al.).
DNMT3B, and DNMT3A mRNA appear to be novel marker for the preferred cell populations according to the invention.
DPPA4
DPPA4 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Markers with On-Off Change in Expression
    Subgroup 1. Glycosyltransferases    Glucosaminyl (N-acetyl) transferase 2 I-branching enzyme GCNT2    UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase polypeptide 3,B3GALT3    CMP-sialic acid alpha2,3sialyltransferase III, ST3GalVI mRNA, SIAT10    Subgroup 2. Nucleotide metabolism enzyme:    Nudix (nucleoside diphosphate linked moiety X)-type motif 5, NUDT5;    Subgroup 3. Glycoprotein:    Synaptic vesicle glycoprotein 2A SV2A    Subgroup 4. Regulatory protein:    Zinc finger protein 117 (HPF9), ZNF117SIAT10
SIAT10 has been reported to have hardly detectable expression in a cultivated hematopoietic cell line (JBC (274) (1999) 11479-86 Okajima T et al.).
SIAT10 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
B3GALT3
Potential product of B3GALT3 globoside has been suggested for signal transduction in embryonal carcinoma cells through AP1 and CREB, no B3GALT3 mRNA expression was indicated in the model (Song Y et al JBC (1998) 273, 2517-25). The relevancy of the data with regard to non-cancer cells of present invention cannot be known with regard to the present invention.
B3GALT3 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
GCNT2
Expression of GCNT2 has been reported from bone marrow CD34+ cells and cultivated differentiating cells thereof (Inaba N et al. Blood (2003) 101, 2870-6). The data does not indicate presence of the mRNA in human cord blood or in any preferred cell population according to the invention. The data does not indicate the usefulness of the marker for differentiation of hematopoietic stem cell types.
GCNT2 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Nudix (Nucleoside Diphosphate Linked Moiety X), NUDT5
NUDT5 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Zinc Finger Protein 117 (HPF9), ZNF117
ZNF117 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Markers Associated with Chromosomal Alterations in Blood Cancers and Other Conditions
Baalc (Brain and Acute Leukemia Cytoplasmic)
Baalc has been reported in another context as development related protein from CD34+ bone marrow cells (Baldus, C D et al. (2003) 31 (11) 1051-6) and associated with mesoderm and muscle development (Satoskar A A et al Gene Expr Patterns (2005) 5 (4) 463-73).
Baalc (brain and acute leukemia cytoplasmic) mRNA appears to be novel marker for the preferred cell populations according to the invention.
T-Cell Lymphoma Breakpoint Associated Target 1, TCBA1
TCBA1 alteration occurs at band 6q21 in T cell lymphomas/leukemias, it may be fusion of TCBA1-SUSP1, or aberrant non-chimeric transcript (Tagaw H. et al. (2002) 34 (2) 175-85)
TCBA1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
Wolf-Hirschhorn Syndrome Candidate 1, WHSC1
This gene is associated with a syndrome caused by deletion of short arm of chromosome 4 associated with a myelo dysplastic syndrome (MDS), possibly caused by allelic loss of WHSC1 (Sharathkuma A et al. Am J Med Genet A (2003) 119 (2) 194-9). In multiple myeloma translocation of t (4; 14) p(16.3; q32) probably deregulated WHSC1 gene (Finelli P et al. Blood (1999) 94 (2) 724-32).
WHSC1 mRNA appears to be novel marker for the preferred cell populations according to the invention, and for stem cells in general.
Antibody Target Structures
The present invention is further directed to cell surface marker structures, which can be recognized by antibodies. This group includes preferred plasma membrane proteins according to the invention, furthermore the invention is directed to molecules characterized by antibodies as cell markers. Preferably this group includes mRNA of serologically defined colon cancer antigen 8, SDCCAG8, and in another embodiment mRNA of Sarcoma antigen NY-SAR-79.
Serologically Defined Colon Cancer Antigen 8, SDCCAG8
SDCCAG8 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Sarcoma Antigen NY-SAR-79
Sarcoma antigen NY-SAR-79 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Islet Cell Autoantigen 1 69kDA, ICA1
ICA1 mRNA appears to be novel marker for the preferred cell populations according to the invention.
Furthermore, the inventors noted a very recent publication (He X et al. (2005) Stem cells and Development 14, 188-198) with a doubly selected cell populations of cord blood. The expression was compared to CD34− cell populations, so the actual relation of the more limited group of markers presented in the article, with regard to preferred cell populations according to the present invention was not indicated. The data appears to be more CD34 related in contrast to the most preferred embodiments of present invention.
The presence of less markers and different markers indicates difference in cell populations. It is also totable that the purification process used by He et al. would not allow to access to present type of cells or corresponding background cell populations
The present invention is not directed to markers of the publication as novel stem cell marker if the markers with similar gene names actually do correspond to the specific markers of the present invention, it is realized that the publication uses different identification number system for the mRNAs.
The present invention is specifically directed to the markers possibly also noted by He et al. when these are analyzed form preferred complete and homogenous cell populations of cord blood selected with regard to single marker according to the present invention.
There is also an increasing need for methods for purification of cell population from various sample types. Purified cell populations are developed for various scientific products and research tools and/or therapeutic products or lead products for therapeutic development.
The samples, or sources of cell populations, are usually tissues, tissue cultures and/or cell cultures. The samples contain beside the target cell population, also other cellular material or other cellular materials. The other cellular material includes multiple different cell populations and/or cell like materials, which should be separated from the desired cell population. The major problems are to maintain the desired cell population intact and remove other cellular material with similarities with the desired cell population. Preferred sample is cell or tissue material containing free or easily mobilizable cells such as blood and/or blood derived materials.
It has been realized that various human tissues contain multipotent cells such as various progenitor cells, or stem cells, which are useful for scientific studies and developing therapeutics for animals and human. There is a need for purification of multipotent cells from various sample types.
The current cell purification methods include affinity methods such as affinity bead methods. The purification methods are not optimal especially for applications requiring highly pure cells. The present invention reveals a novel purification method, which allows effective purification of a cell population. The method yields especially complete cell populations. The complete cell populations are especially useful for scientific development, diagnostics development and cell culture and development thereof.
Hematopoietic stem cells (HSC), with their unique self-renewal and differentiation capacity, offer great potential for the treatment of hematological disorders, immunodeficiency and inborn errors of metabolism [51, 52]. HSCs can be collected from mobilized peripheral blood (PB), bone marrow (BM) and cord blood (CB). Lately CB has been increasingly utilized because it is readily available, HLA mismatch is better tolerated and there is a decreased risk of graft-versus-host disease when using CB-derived HSCs [53]. Even though the cell content of CB is limited, it has higher frequency of progenitors compared to PB or BM [53-55]. CB-derived CD34+ cells have also been shown to proliferate more rapidly than their counterparts from BM [56], and CB-derived HSCs possess increased engraftment potential when compared to cells from PB or BM [57, 58]. In addition, recent studies suggest that CB is a source of non-hematopoietic stem cells [59, 60].
Enrichment of HSCs is based on the surface expression of phenotypic markers or the lack of expression of lineage-specific markers. The most commonly used surface marker for HSC selection is CD34, a transmembrane glycoprotein expressed on HSCs. CD34 is also used to quantify the stem cell content of collected units in CB banks [61]. Most, if not all, CD34+ cells express CD133 glycoprotein on their surface. CD133 is appears to be expressed on more primitive cells and CD133+ grafts have been tested in stem cell transplantation [62-64]. Fluorescence-activated cell sorting and immunomagnetic selection system employ these cell surface antigens to enrich HSCs. However, a major challenge has been the difficulty to produce highly pure HSC fractions with good recovery. Furthermore, the handling of CB is challenging due to the relatively high content of thrombocytes and nucleated erythroid precursors. For these reasons, standardized protocols for PB sample handling and cell separation do not work well for CB. Only few studies have investigated the efficiency of the immunomagnetic selection method used to isolate CD34+ cells from CB. Belvedere et al. compared the results from 49 selections and reported mean CD34+ cell purities of 41% and 85% after first and second passage through the separation column, respectively [67]. Melnik et al. report an average purity of 60% for CB-derived CD34+ cells from 10 separations [68].
In this invention, different protocols were optimized to enrich HSCs with over 90% purity from fresh and cryopreserved CB. Cryopreserved CB cells have been considered especially challenging in selection procedures because of cell aggregation caused by cell damage during thawing. The used protocols were based on positive selection of cells expressing CD34 and CD133. The magnetic cell sorting system MACS was used due to its gentleness and time-effectiveness. Further, the clonogenic capacity of selected HSC populations was determined using colony-forming unit (CFU) assay.